P1433: USE OF A FOAMY-VIRUS VECTOR SYSTEM TO PRODUCE AN ‘OFF-THE-SHELF’ ANTI-CD19 CAR T CELL PRODUCT
Authors: I. Lazana, E. Simantirakis, M. Daniil, I. Tsironis, G. Vassilopoulos
Journal: HemaSphere (2022)

Background: Anti-CD19 chimeric antigen receptor (CAR) T cells have transformed the field of cancer immunotherapy. Current CAR T cell trials use autologous cells, with several limitations (prolonged production time, costly, manufacturing failure), whereas the Lentiviral vectors (LV), used for commercial products, are endowed with extra limitations (packaging limits and mutagenesis risk). Aims: Our group has developed an in-house anti-CD19 CAR-T cell product, using a safer to LV, foamy virus vector (FV). Methods: FV vectors expressing the anti-CD19 CAR from an EFP1 promoter were constructed. 2nd generation LV vector backbones were purchased from a commercial vendor. Peripheral blood from healthy individuals were used as T cell sources. T cells were activated by antiCD3/CD28 beads and transduced with antiCD19 CAR-T, LV or FV vectors. Transduction efficiency was assayed by flow cytometry (FCM). FV and LV CAR-T cells were expanded with a modified Rapid Expansion Protocol (REP). Immunophenotypic analysis of CAR T cells was performed by FCM. Their cytotoxic effect was evaluated against the CD19+ Raji and Daudi cell lines and against the CD19- cell line, HL60. Cytotoxicity was assessed by FCM and calculated as: [1-live targets (sample)/live targets (control)]x100. CAR-T cell activation was also assayed by INF-γ ELISA. Results: LV and FV vector titers were between 3-5x10^5 TU/ml and 4-5x10^5 TU/ml, respectively. Transduction efficiency ranged from 45-83% at MOI 3-5 with FV vectors and was comparable to the transduction efficiency of LV vectors at a much higher MOI (10-20). Following REP, the vast majority of cells consisted of CAR T cells, regardless of the vector used or the T cell source and isolation method. Immunophenotypic analysis of CAR T cells revealed a central memory phenotype (CD45RO+/CD45RA-/CCR7+), (>99% cells). For cytotoxicity assays, CAR-T cells were incubated with CFSE-labelled Raji and/or Daudi cells at different ratios (5:1, 10:1) for 18 hours. At the end of the incubation period, the % cell lysis was 87.3 (SD 6.38) and 92.4 (SD 3.2) at 5:1 and 10:1 ratio, respectively (n=3). Similar results were obtained for LV vectors. When CD19- HL60 cells were used as targets, no/minimal lysis was noted, indicating a specific anti-CD19 cytolytic effect of CAR T cells. IFN-γ levels were measured at the end of the co-culture period in the supernatant to assess CAR T cell activation. Results showed a significant increase both in the FV- and LV- CAR T cells, compared to untransduced cells (mean 890.2 and 3,141pg/ml and 1,553 and 2,225pg/ml for FV- and LV- CAR T cells, in ratios 5:1 and 10:1, respectively). Integration site analysis of FV vectors in the T-cells is under way. Summary/Conclusion: Our group has developed for the first time a FV vector for anti-CD19 CAR T cell production, with an efficient gene transfer to human T cells and with potent in vitro cytotoxic properties, similar to their LV-derived counterpart. Overall, we provide a proof of concept that allogeneic, in-house CAR T cells derived from an alternative viral backbone such as the FV, could be a safe, efficient and affordable vehicle to LV-derived vectors for immunotherapy.